Molecular Sciences Isolation and Characterization of Microsatellite Loci for Hibiscus Aridicola (malvaceae), an Endangered Plant Endemic to the Dry-hot Valleys of Jinsha River in Southwest China

Hibiscus aridicola (Malvaceae) is an endangered ornamental shrub endemic to the dry-hot valleys of Jinsha River in southwest China. Only four natural populations of H. aridicola exist in the wild according to our field investigation. It can be inferred that H. aridicola is facing a very high risk of extinction in the wild and an urgent conservation strategy is required. By using a modified biotin-streptavidin capture method, a total of 40 microsatellite markers were developed and characterized in H. aridicola for the first time. Polymorphisms were evaluated in 39 individuals from four natural populations. Fifteen of the markers showed polymorphisms with two to six alleles per locus; the observed heterozygosity ranged from 0.19 to 0.72. These microsatellite loci would be useful tools for population genetics studies on H. aridicola and other con-generic species which are important to the conservation and development of endangered species.


Introduction
Genus Hibiscus (Malvaceae) includes at least 250 species distributed in tropical and subtropical regions [1]. H. aridicola is endemic to the dry and hot valleys of the Jinsha River (upper reaches of the Yangtse River) in Southwest China. It is an ornamental deciduous shrub with large flowers that vary from white to whitish purple. Although the species was described in 1927 [2], and literature or relative research references are limited. H. aridicola was widely distributed along the valleys of Jinsha River from 1300 m to 2100 m during the 1950s [2][3][4]. However, it currently has been evaluated as Endangered [EN B2ab (ii)] (following IUCN Red List Categories and Criteria) with less than five locations and a continuing decline of area of occupancy [5]. Our recent field surveys have further confirmed the threatened status of the species and only four natural populations of H. aridicola exist in the wild. Our surveys have also discovered that habitats of H. aridicola have been greatly degraded and the populations are fragmented and isolated from each other. Population genetic studies have predicted that fragmentation will lead to a loss of genetic diversity due to inbreeding, population isolation and restricted gene flow and small effective population sizes and that may lead to a decline in fitness or even, ultimately, extinction [6,7]. As an endangered and endemic ornamental shrub, H. aridicola is facing a very high risk of extinction in the wild, and its effective and long-term conservation is urgently needed. In our study, 40 microsatellite markers were developed and characterized to investigate the genetic diversity and population structure using the fast isolation by Amplified Fragment Length Polymorphism (AFLP) of sequences containing repeats (FIASCO) [8].

Results and Discussion
A total of 441 positive clones were captured, among these 232 clones (53%) were found to contain simple sequence repeat (SSR). Finally, 84 sequences contained SSR loci were selected for primer design. 40 microsatellite loci successfully amplified in H. aridicola. We tested the degree of polymorphism in four populations of H. aridicola for 40 microsatellite loci and 15 out of them are polymorphic amplification, other 25 microsatellite loci were monomorphic by the result of polyacrylamide gel (Table 1).

Population 1 (N = 10) Population 2 (N = 10) Population 3 (N = 10) Population 4 (N = 9)
Locus These microsatellite markers developed in our study will be a useful tool for further studies of conservation genetics, and will help us understand the genetic structure of H. aridicola, so as to make effective conservation strategy for this endangered plant.

Experimental Section
Total genomic DNA of H. aridicola was extracted from dry leaf tissue of three different individuals using a modified 4 × CTAB method [9]. A microsatellite enriched library was conducted by using a modified biotin-streptavidin capture method. Firstly, the genomic DNA (about 800-1000 ng) was completely digested with MseI restriction enzyme (NEB) into the sequences between 200 bp-800 bp, then the digested fragments were ligated to MseI AFLP adaptor following by amplification with adaptor-specific primers (5'-GAT GAG TCC TGA GTA). After a PCR the products were hybridized with biotinylated probes (AG) 15 , (AC) 15 and (AAG) 10 respectively [8]. The PCR products were ligated into PGEM-T vector (Promega) after purified, and transferred into E. coli strain DH5α (Tiangen). The proportion of products and PGEM-T vector in the total volume should be controlled between 3:1 and 5:1. Positive clones were picked out and tested using (AG) 10 /(AC) 10 /(AAG) 7 primers and vector primers SP6/T7 respectively to select appropriate fragments which contained SSR. The positive clones were captured for sequencing with an ABI PRISM 3730XL SEQUENCER. Sequences contained simple sequence repeat were selected for primer design using Primer Premier 5.0 program [10].
The designed Primer pairs were tested in 39 wild H. aridicola samples from four populations in Southeast China. Herbarium voucher deposited in Kunming Institute of Botany, Chinese Academy of Sciences (code ZL0017-0019, ZL0108-ZL 0120). The PCR amplification was carried out in a total volume of 10 μL reaction containing 5 μL 2 × Taq PCR MasterMix (Tiangen; 0.1 U Taq Polymerase/μL, 0.5 mM dNTP each, 20 mM Tris-HCl (Ph8.3), 100 mM KCl, 3 mM MgCl 2 ), 0.5 μL of each primer and 0.5 μL Genomic DNA. Amplification was carried out in thermocycler (veriti 96 well thermal cycler) with a cycling profile 95 °C for 4 min then 35 cycles of 45 s at 94 °C, 45 s at the specific annealing temperature, 45 s at 72 °C and a final extension step of 8 min at 72 °C. The amplification products were separated on 8% denaturing polyacrylamide gels and visualized by silver staining with a 100 bp extended DNA ladder (Fermentas) as a size standard.
The data was analyzed by GENEPOP 4.0 [11], which included test of observed heterozygosity (H O ) and expected heterozygosity (H E ) for the 15 polymorphic microsatellite loci.

Conclusions
In summary, 40 microsatellite markers have been specifically developed for H. aricicola in this study. The high discriminatory power of 15 polymorphic loci suggests that they should be suitable for the fine-scale analysis and survey of population structure in scarce populations of the endangered H. aridicola. These developed and characterized SSR markers for H. aridicola would also be useful for exploring genetic diversity and genetic structure of other species in Hibiscus.